In this research project, we have investigated how modification of macrophages rather than modification of LDL affects macrophage metabolism of native LDL. We have learned that macrophage cholesterol accumulation can occur with native LDL when macrophages are differentiated from monocytes with the specific growth factor, M-CSF. M-CSF differentiated macrophages show constitutive uptake and degradation of native LDL. LDL uptake does not depend on macrophage oxidation of LDL or macrophage binding of LDL. Rather, the M-CSF differentiated macrophages take up LDL by constitutive fluid-phase pinocytosis, a process by which macrophages and some other cell types take up large amounts of fluid. The macrophages take up LDL as part of the fluid that they ingest during pinocytosis. This produces cholesterol accumulation in macrophages to levels characteristic of macrophage foam cells in atherosclerotic plaques. This novel mechanism of macrophage cholesterol accumulation shows that oxidative modification of LDL is not necessary for foam cell formation to occur. Human M-CSF differentiated macrophages, the predominant macrophage phenotype that we showed previously in human atherosclerotic plaques, constitutively take up native LDL by fluid-phase pinocytosis. Fluid-phase pinocytosis is a cellular mechanism of receptor-independent material uptake that occurs by the formation of large vacuoles (>0.5 microns) through a process called macropinocytosis, and/or formation of small vesicles (<0.2 microns) through a process called micropinocytosis. Macropinosomes form by actin-rich plasma membrane protrusions extending from the cell and subsequently fusing with the plasma membrane, resulting in the engulfment of large volumes of fluid. In contrast, micropinosomes form by membrane invagination and scission to form small vesicles. By these mechanisms of uptake, material present within fluid (such as LDL) is taken up by cells at levels that are directly proportional to the concentration of the material present within the fluid. In contrast to receptor-mediated uptake of material, uptake of material by fluid-phase pinocytosis does not require receptors to bind the material and therefore does not show receptor saturation. Consequently, when concentrations of material are high, similar to LDL present within the vessel wall, substantial cellular uptake of this material occurs. This past year, we have examined the contribution of macro- and micropinocytosis pathways to fluid-phase LDL uptake by macrophages, and have begun to elucidate what drugs can inhibit these uptake pathways. We identified the kinase inhibitor SU6656 and the Rho GTPase inhibitor toxin B as inhibitors of macrophage fluid-phase pinocytosis of LDL. Assessment of macropinocytosis by time-lapse microscopy revealed that both drugs almost completely inhibited macropinocytosis, although LDL uptake and cholesterol accumulation by macrophages was only partially inhibited (about 40%) by these drugs. We therefore investigated the role of micropinocytosis in mediating LDL uptake in macrophages and identified bafilomycin A1 as an additional partial inhibitor (about 40%) of macrophage LDL uptake that targeted micropinocytosis. When macrophages were incubated with both bafilomycin A1 and SU6656, inhibition of LDL uptake was additive (reaching 80%), showing that these inhibitors target different pinocytosis pathways. Microscopic analysis of fluid-phase uptake pathways in these macrophages confirmed that LDL uptake occurs through both macropinocytosis and micropinocytosis. Our findings show that human M-CSF-differentiated macrophages take up native LDL by macropinocytosis and micropinocytosis, underscoring the importance of both pathways in mediating LDL uptake by these cells. In conclusion, our findings show that fluid-phase pinocytosis of LDL is a plausible mechanism that can explain how macrophages accumulate cholesterol and become disease-causing foam cells. The findings direct attention to macrophage pinocytosis as a relevant pathway to target for modulating macrophage cholesterol accumulation in atherosclerosis. Our research seeks to identify additional drugs that inhibit macrophage pinocytosis of LDL, and thereby may inhibit the development of atherosclerotic diseases of blood vessels.